Investigating body function

ABSTRACT

Body function of a mammal is investigated using a γ-emitting  75  Se or  123m  Te labelled derivative of a bile acid or bile salt. Thus bowel function may be investigated by oral administration of the labelled compound followed, after a suitable period of time, by either a whole body count or a faecal count of radioactivity. Preferred bile acids and salts are substituted either at the 19-position or in the C-17 side chain.

This invention relates to the investigation of body function, especiallysmall bowel function but also liver function, using bile acids and bilesalts or their metabolic precursors labelled with radio isotopes ofselenium or tellurium.

Bile salts are synthesized in the liver from cholesterol, pass via thehepatic and common bile ducts to the intestinal tract, are reabsorbed inthe ileum and return to the liver via the portal venous system. Duringthis enterohepatic circulation in a normal human more than 95 percent ofthe bile salts entering the small intestine are reabsorbed, theremainder entering the large intestine and eventually appearing in thefaeces. Malfunctioning of the ileum, which can be caused by a number ofpathological conditions, can result in the deficient absorption of bilesalts. A measurement of bile salts absorption by the intestine wouldtherefore provide useful information enabling the distal small bowel tobe recognized, or eliminated, as the source of gastrointestinaldisorder.

Bile acids may be represented by the following formula: ##STR1## whereinR₁, R₂,R₃ and R₄ are, independently a hydrogen atom or an α- orβ-hydroxyl group, and wherein H₅ is either in the α or β position.

Bile salts are conjugates of the above bile acids with amino acids, inparticular glycine and taurine.

Carboxyl-¹⁴ C-cholic acid (1, R₂ =H, R₁ =R₃ =R₄ =α-OH; H₅ is β) and itstaurine conjugate have been used to study the absorption of bile saltsin the intestine of both animals and man under a variety of pathologicalconditions, e.g. regional ileitis, ileal resection, and induceddiarrhoea. The investigations have required the measurement of ¹⁴ Cradioactivity in faeces, urine and bile. In the breath test as devisedby Fromm and Hofmann glycine-1-[¹⁴ C]glycocholate is used to detectincreased bacterial deconjugation of the bile salts. Upon deconjugationin the small bowel as a result of bacterial overgrowth or in the colonfollowing bile salt malabsorption, the glycine liberated is metabolized,absorbed, and partly exhaled as ¹⁴ CO₂. In the case of bile saltmalabsorption some of the ¹⁴ C radioactivity will appear in the faeces.A faecal ¹⁴ C measurement is essential for complete exploitation of thediagnostic scope of the breath test. In the diagnosis of bile acidmalabsorption the Schilling test employing labelled cyanocobalamin withintrinsic factor is often helpful, but by itself it cannot discriminatebetween bacterial overgrowth and ileal dysfunction.

The measurement of bile acid adsorption as a routine test for smallbowel function would be greatly facilitated if the bile acids could belabelled with a gamma emitting isotope. Counting of gamma emitters is ingeneral easier and more economical than is counting of beta emitters:this particularly applies to biological samples such as bile or faeces,where for beta emitters it would be necessary to process the samplebefore counting could begin. Labelling with a gamma emitter wouldpossess the additional advantage of allowing body counting and thusobviate the need to handle faecal samples; visualisation of theenterohepatic system would also be possible. The gamma emitting isotopeswhich could possibly be employed to label bile acids without changingtheir biological behaviour, and in which the label would remain attachedthroughout the enterohepatic cycle, are limited in number. Thisinvention concerns the use of radioisotopes of selenium and tellurium,such as selenium-75 and tellurium-123m, to fulfil the required function.The incorporation of either selenium or tellurium into the structure ofthe bile acid molecule has so far not been described; this applies toboth the radioactive and non-radioactive forms of these elements.

The idea behind this invention arises from the observation that when19-methyl-[⁷⁵ Se]selenocholesterol was administered intravenously tohumans with the object of adrenal visualisation the selenium-75radioactivity became concentrated in the enterohepatic system and thatthe metabolism of 19-methyl-[⁷⁵ Se]selenocholesterol appeared toparallel that of cholesterol. It was surmised that 19-methyl-⁷⁵ Seselenocholesterol was being converted to analogues of bile salts inwhich a methylseleno group was attached to the C₁₉ carbon atom.

The present invention provides, in one aspect, a method forinvestigating body function, especially small bowel function, of amammal, comprising introducing a γ-emitting radioactive Se or Telabelled derivative of a bile acid or an amino acid conjugate thereof(bile salt) or a metabolic precursor thereof into the live mammal, andafter the elapse of a suitable period of time determining thedistribution of the radioactivity. For determining small bowel function,the labelled bile acid or bile salt is preferably introduced orally. Thedistribution of radioactivity may be determined by counting of bodyradioactivity, e.g. by means of a whole body counter, or by measurementof faecal radiation. Where faecal measurements are used this inventionincludes the use of a second, non-absorbable, γ-emitting isotope, e.g.¹³¹ I-polyvinylpyrollidone or ⁵¹ Cr as a marker. This investigation ofbody function may involve visualising a part, e.g. the hepatobiliarysystem, of the mammal, by introducing in the live mammal a γ-emittingradioactive selenium or tellurium derivative of a bile acid or its aminoacid conjugate, allowing the labelled bile acid to concentrate in thepart, e.g. the hepatobiliary system, and observing the radiation emittedby the labelled bile acid in the said part.

In the case of whole body and faecal determination of Se or Tedistribution, measurements cannot commence until after excretion of someof the radioactivity from the body. This would normally be a minimum ofabout 24 hours after ingestion of the labelled bile salt but could varywith some patients. It is also possible to carry out measurements overan extended period of time, say over 7-14 days, in order to gain moreaccurate estimates of excretion rates. In the case of plasmadetermination measurements may be started in less than 24 hours afteringestion of the bile salt since absorption through the intestine intoplasma would occur well before elimination of radioactivity via thefaeces. Here again it is also possible to carry out measurements over anextended period of time in order to gain more accurate information.

It has recently been shown that the blood level of bile acids and bilesalts, determined in vitro by radioimmunoassay, can provide a sensitiveindication of liver function. According to the present invention, theintroduction of a radioactively labelled bile acid or bile salt into thebloodstream enables this determination to be effected in vivo quicklyand simply. For this purpose, the labelled bile acid or bile salt ispreferably administered intravenously, and samples of blood taken forradioactive counting after appropriate intervals of time.

When the mammal is an adult human being, the dose administered isgenerally in the range of 1 to 500 μCi, e.g. from 1 to 50 μCi forinvestigating organ function or from 50 to 500 μCi for organvisualisation.

Techniques for introducing a bile acid into live mammals and allowing itto become absorbed and localized are known in the art and will not befurther described here. Measurement of the γ-radiation emitted by theselenium or tellurium and visualization of the hepatobiliary system orother parts of the mammal where the labelled bile acid is concentrated,can be effected with standard equipment. Equipment for measuring bodyradioactivity may suitably be a whole body counter, or a γ-camera withthe collimator removed which may be pointed at all or just the relevantpart of the body.

The labelled bile acids or their salts and their metabolic precursorsused in the above method include those shown in formulae (2) and (3)below and which are substituted by either Se or Te either at the C-19position or in the C-17 side chain of the molecule; also Se or Telabelled derivatives of compounds such as7α-hydroxy-19-methylselenocholesterol which are intermediate in themetabolic conversion of compounds such as 19-methylselenocholesterol toa 19-methylseleno bile acids or bile salts; also those shown in formula(4) below. ##STR2## wherein Z is a γ-emitting radioactive isotope ofselenium or tellurium,

R¹ =alkyl (e.g. C₁ -C₄ alkyl); cycloalkyl (e.g. cyclohexyl); or aralkyl(e.g. benzyl)

R², R³, R⁴, and R⁵ are independently hydrogen or an α- or β-hydroxylgroup,

R⁶ is --OH or an amino acid residue

H⁵ may be α-H or β-H

n=0 or 1, ##STR3## wherein R is ##STR4## and A is 0 or 1,

B is 0 to 4,

C is 0 to 4,

Z is a γ-emitting radioactive isotope of selenium or tellurium,

R⁶ is --OH or an amino acid residue,

R⁷ is hydrogen or saturated C₁ to C₄ alkyl group when A is 1,

R⁸ is hydrogen or saturated C₁ to C₄ alkyl group,

n is 0 or 1,

R², R³, R⁴ and R⁵ are independently hydrogen or an α- or β-hydroxylgroup, or an oxo group,

H₅ is an α- or β-H. ##STR5## wherein R⁶ is --OH or an amino acidresidue.

R⁹, R¹⁰, R¹¹ and R¹² are independently a hydrogen atom, an α- orβ-hydroxyl group or an α- or β-SeCH₃ group, -SeR¹³ where R¹³ is C₁ to C₄alkyl, provided that at least one is an α- or β-group -SeR¹³, and

H₅ is either in the α- or the β- position.

The present invention provides, in a second aspect, labelled bile acidsand their bile salt derivatives substituted by Se or Te at either 19C orin the C-17 side chain and defined by the formula (2) and (3) aboverespectively.

This invention includes the inactive compounds and also, moreparticularly, the compounds labelled with radioactive isotopes ofselenium and tellurium, e.g. selenium-75 and tellurium-123m. Theinactive compounds are useful aids in determining the properties of theradioactive compounds.

The labelled bile acids of the present invention and their amino acidconjugates may be prepared by the following routes:

1. 19-alkylseleno bile acids and their conjugates

The compounds of this group corresponding to formula (2) may be preparedbiologically.

19-methyl-[⁷⁵ Se]selenocholesterol, or a suitable intermediate on themetabolic pathway of its conversion to a bile salt analogue, e.g.7α-hydroxy-19-methyl-[⁷⁵ Se]selenocholesterol, is administeredintravenously to a suitable animal, e.g. rat or rabbit, with a biliaryfistula. The ⁷⁵ Se compound is converted in the liver of the live animalto a ⁷⁵ Se-labelled bile salt analogue, which is controlled in the bilevia the biliary fistula. Methods of separating and purifying bile acidsand their conjugates from samples of bile are known in the art. Forexample, the bile is added to 20 volumes of absolute ethanol. Afterbrief boiling and subsequent cooling the extract is filtered andevaporated to dryness. The solid residue after extraction with petroleumether to remove fats, is then dissolved in methanol and the methanolicsolution, after filtration to remove inorganic salts, is evaporated todryness in vacuo to yield a mixture of natural and ⁷⁵ Se-labelled bilesalts. The crude product may be further purified by preparativethin-layer chromatography. The advantage of using an intermediatemetabolite such as 7α-hydroxy-19-methyl-[.sup. 75 Se]selenocholesterolis that higher yields of the ⁷⁵ Se bile acids are obtained. An isolatedperfused liver may be used as an alternative to using a live animal forthe biological preparation of a ⁷⁵ Se-labelled bile salt.

The above described process has been used to provide analogues of bilesalts in which the methyl group attached to the C₁₀ carbon atom isreplaced by a methylselenomethyl group. The products behaved as aminoacid conjugates of bile acids both in their chromatographic behaviourand as substrates for the enzyme cholylglycine hydrolase.

2. BILE ACIDS AND THEIR CONJUGATES WITH A SELENIUM ATOM IN THE C₁₇ SIDECHAIN

The compounds of this group, corresponding to formula (3), may beprepared by the reaction of a suitable selenium or tellurium nucleophilewith a modified bile acid having a terminal halogen atom, e.g. bromineor iodine, in the C₁₇ side chain. These reactions are carried out insuch solvents as ethanol, propanol, tetrahydrofuran ordimethylformamide, or mixtures of these solvents, generally at roomtemperature. The selenium or tellurium nucleophiles are produced by thereaction in liquid ammonia of disodium diselenide or ditelluride with anω-halogenated carboxylic acid or its ester, the resulting organicdiselenide or ditelluride being dissolved in one of the above solventsand cleaved by reagents such as sodium borohydride or dithiothreitol;further reaction with the modified bile acid is effected in situ.

Alternatively, the halogenated bile acid may be reacted with disodiumdiselenide in a solvent such as propanol at elevated temperatures toprovide a disteroidal diselenide. The disteroidal diselenide isdissolved in ethanol, cleaved with sodium borohydride, and the selenolreacted in situ with an ω-halogenated carboxylic acid ester.

The use of potassium selenocyanate affords a useful route to thecompounds of this group. Potassium selenocyanate, prepared by dissolvingred selenium in ethanolic potassium cyanide, is reacted in ethanol atreduced temperatures with a ω-halogenocarboxylic acid ester. Theresulting ω-selenocyanato-carboxylic acid ester is reduced with sodiumborohydride and reacted in situ with the halogenated bile acidintermediate. These reactions are usually conducted at room temperaturein ethanol or ethanol/tetrahydrofuran mixtures.

ω-Halogenated carboxylic acid esters may be used in place ofω-halogenated compounds in the above reaction to provide products havinga side chain in the α-position to the carboxyl group.

Where hydroxyl groups have been protected by acylation and carboxylicacid groups by esterification the protecting groups are removed bystandard methods prior to final purification of the product bypreparative layer chromatography on silica gel.

The bile acid analogues, containing either a selenium or a telluriumatom in the C₁₇ side chain, may be conjugated via an amide linkage toamino acids such as glycine and taurine. The methods used to prepare thebile acid conjugates are well known in the art and depend on thecondensation of the bile acid with the amino acid in a suitable solventsuch as dimethylformamide and in the presence of a condensing agent suchas a carbodiimide or N-ethoxy-carbonyl-2-ethoxy-dihydroquinoline (EEDQ).

The reaction schemes below illustrate the preparations broadly describedabove. Further detail is provided in the Examples. It is to beunderstood that these preparations may be carried out with eithernatural selenium or tellurium or with these elements enriched with theirrespective radioactive isotopes, e.g. ⁷⁵ Se or ^(123m) Te. ##STR6## N.B.R=bile acid nucleus

X=halogen

The insertion of either a selenium or tellurium atom into the C₁₇ sidechain of a bile acid according to formula (3) is dependent upon theavailability of modified bile acid intermediates having a terminalhalogen atom, e.g. bromine or iodine, in the C₁₇ side chain. Theprovision of such intermediates has required the shortening orlengthening of the C₁₇ side chain by methods known in the art, e.g.Barbier-Wieland degradation or the Arndt-Eistert reaction respectively.Replacement of the terminal carboxyl group by a halogen atom may beeffected by the Hunsdiecker reaction. A particularly effective means ofaccomplishing this replacement is to treat the bile acid in refluxingcarbon tetrachloride with lead tetra-acetate/iodine reagent, thereaction mixture being irradiated with light meanwhile. The hydroxylgroups of the bile acid must be protected with suitable groups such asformyl, acetyl, nitro, etc. This reaction results in the replacement ofthe carboxyl group with an iodine atom. The degradation of the C₁₇ sidechain of cholic acid to provide a 20-iodo-5β-pregnane derivative may beeffected by three consecutive reactions:

(1) refluxing of the protected bile acid in dry benzene and undernitrogen with lead tetraacetate in the presence of cupric acetate andpyridine provides a corresponding Δ²² -24-nor-5β-cholene, (A);

(2) treatment of A with sodium periodate/potassium permanganate inaqueous 2-methylpropan-2-ol in presence of potassium carbonate causesoxidation of the Δ²² bond and provides the3α,7α,12α-triformoxy-23,24-bisnor-5β-cholanic acid, (B);

(3) B is refluxed in carbon tetrachloride with lead tetraacetate/iodinereagent under light irradiation to provide the3α,7α,12α-triformoxy-20-iodo-5β-pregnane derivative, (C). C is probablya mixture of R and S isomers but the proportions have not beendetermined.

The above reaction can be performed equally using steroids in the 5α- orthe 5β- configuration. Available steroids in the 5α- configurationinclude 5α-cholanic acid-3β-ol and 22,23-bisnor-5α-cholanic acid-3β-ol.

3. BILE ACIDS WITH RING SELENOALKYL SUBSTITUTION

The compounds of this group, corresponding to formula (4) may beprepared from the corresponding ring-OH substituted compound by firstreplacing hydroxyl by iodine and then replacing iodine by selenoalkyl inknown manner.

By means which are described in general terms above and in detail in thepreparative Examples below, the following ten compounds were prepared:

COMPOUNDS PREPARED (DEFINITIVE NOMENCLATURE)

(i) 19-methyl-[⁷⁵ Se]seleno bile salts prepared biosynthetically from19-methyl-[⁷⁵ Se]selenocholesterol.

(ii) 3α,12α-dihydroxy-22-(carboxymethyl-[⁷⁵Se]seleno)-23,24-bisnor-5β-cholane

(23-[⁷⁵ Se]seleno-25-homodeoxycholic acid)

(iii) 3α,7α-dihydroxy-23-(β-carboxyethyl-[⁷⁵Se]seleno)-24-nor-5β-cholane

(iv) 3α,7α,12α-trihydroxy-23-(β-carboxyethyl-[⁷⁵Se]seleno)-24-nor-5β-cholane

(v) 3α,7α,12α-trihydroxy-20-(carboxymethyl-[⁷⁵ Se]seleno)-5β-pregnane

(22-[⁷⁵ Se]selenacholic acid)

(vi) Glyco-22-[⁷⁵ Se]selenacholic acid

(Glycine conjugate of v)

(vii) 3α-hydroxy-24-(carboxymethyl-[⁷⁵ Se]seleno)-5β-cholane

(viii) 3α,12α-dihydroxy-23-(carboxymethyl-[^(123m)Te]telluro)-24-nor-5β-cholane

(ix) Tauro-23-[⁷⁵ Se]selena-25-homodeoxycholic acid

(Taurine conjugate of ii)

(x) 3,7,12-triketo-23-(carboxymethyl-[⁷⁵ Se]seleno)-24-nor-5β-cholane.

N.B. When selenium and tellurium are used as part of a prefix e.g.carboxymethylseleno, the words used are seleno and telluro. Selena andtellura are used to denote replacement of a CH₂ group e.g.22-selenacholic acid denotes replacement of the C₂₂ carbon atom by Se.

BIOLOGICAL EVALUATION A. Tissue Distribution Studies

Tissue distribution studies in rats, after oral administration ofcompounds i to viii, show that these compounds are excreted in thefaeces to an extent greater than 90 percent after a period of 7-8 days.The pattern of tissue distribution is similar for all the compounds. At20 hours the ⁷⁵ Se radioactivity is largely confined to the liver, gutand faeces; apart from compounds iii and iv less than 5 percent of theradioactivity is distributed among other organs. Compound ii at thistime exhibits a somewhat different behaviour in that it shows a verymuch small percentage of ⁷⁵ Se radioactivity in the faeces and acorrespondingly larger proportion in the small bowel.

B. Whole-Body Excretion Studies

Whole-body excretion studies in rats over a period of 8 days after oraladministration of compounds i to viii indicate that excretion is delayedwith respect to a non-absorbable radioactive maker, ¹³¹I-polyvinylpyrollidone.

For each of the eight compounds under investigation, approximately 10-15μCi was administered via an intragastric tube to rats. Whole-body countswere determined immediately after administration and at intervals duringthe following 6-8 days. A whole-body standard permitted corrections forradioactive decay and variations in counter efficiency.

Whole-body retention is expressed as a percentage of the countsdetermined immediately after administration.

Graphical analysis of the results revealed that, in every case, the datacould be approximated by a function of the form:

    Retention (t)=ae.sup.-xt +be.sup.-yt +c                    (1)

The values obtained for each of the parameters in equation 1 for each ofthe substances are tabulated below.

    ______________________________________                                        a(%)        x(d.sup.-1)                                                                            b(%)     y(d.sup.-1)                                                                           c(%)                                    ______________________________________                                        ii     190      2.29     27.6   0.65    3.36                                  iv     260      2.77     42.5   0.66    15.5*                                 iii    170      2.31     37.5   0.53    4.0                                   v       98      2.52     16.5   0.38    0.8                                   vi     221      1.54     7.2    0.54    0.58                                  vii    203      2.29     11.8   0.44    2.85                                  i       80      2.02     7.1    0.62    6.0                                   viii   173      2.26     6.3    0.43    1.3                                   ______________________________________                                         *Probably closer to 5.5% as revealed by dissecton results.               

The first component (ae^(-xt)) is variable. A consistent pattern emergeshowever. The half-life of this first component ranged from 0.25 to 0.45days. The half-life of the non-absorbed gastrointestinal marker, I¹³¹P.V.P. has been shown to be approximately 0.17 days, and thus the firstcomponent of the bile salt excretion does not represent material thathas passed unabsorbed through the alimentary canal. It may howeverrepresent that portion of the administered bile salt that afterabsorption, is metabolised on its first pass through the liver, and isnot reabsorbed.

The second component (be^(-yt)) had a half-life in the range 1.05 to1.82 days. This is approximately 10 times the half-life of anon-absorbed marker and certainly represents material that has beenabsorbed from the gastrointestinal tract. This material is probablyincorporated into the enterohepatic circulation.

The constant (c) represents that percentage of the administeredsubstance that is predicted mathematically as never being excreted (orexcreted with a very long half-life compared to those of the othercomponents). The figure for the tri-hydroxy compound isanomalous--tissue distribution studies at the end of 8 days indicated awhole body retention of approximately 5.5%.

C. Biliary Excretion Studies

The biliary excretion from rats of ¹⁴ C/⁷⁵ Se radioactivity after oraladministration of a mixture of a ⁷⁵ Se-seleno bile acid and ¹⁴ C-cholicacid provides information not revealed in the previous studies, The ¹⁴C-cholic acid acts as an internal comparison marker for each ratstudied. Measurement of the ratio of the ¹⁴ C/⁷⁵ Se radioactivitycollected in a 24 hour bile sample to the ¹⁴ C/⁷⁵ Se radioactivityadministered orally gives an indication of the efficiency of absorptionof the seleno bile acid as compared to ¹⁴ C-cholic acid. Ideally, whenthese absorptions are the same the ratio would be 1, but as theefficiency of the adsorption seleno bile acid diminishes the ratioincreases. In the compounds studied the ratio for i,ii and v ranges from3 to 5, whereas for iii it is 54 which indicates a very much reducedabsorption. In the case of compounds i, ii and v the pattern ofappearance of ⁷⁵ Se radioactivity in the bile is similar to that of the¹⁴ C radioactivity. This similarity, which is not shown by compound iii,indicates the same site of absorption for both the seleno bile acid andcholic acid.

D. Clinical Use

It has been shown by whole-body counting that in normal humans having nopast history of bowel dysfunction the rate of elimination of ⁷⁵ Se-iifrom the body is considerably slower than it is for rats. However, inhumans who have suffered on ileal resection this rate of elimination isincreased.

These ⁷⁵ Se-seleno bile acids may therefore be used to investigatemalabsorption of bile acids associated with ileal dysfunction.Measurement of rate of excretion may be performed either by whole-bodycounting or by faecal counting utilising a γ-scintillation counter.

CLINICAL EXAMPLES

Approximately 1 μCi of compound ii was given orally as a drink in waterto four adult volunteers, two of them being normal active adult males,35-50 years of age, and two who had received a total ileal resectionmore than 10 years previously. All were on a normal diet. Se-ii wasadministered between 10.00 am and 12 noon. Body radioactivity was thenmeasured in a whole-body counter immediately after administering the ⁷⁵Se-ii and subsequently at 48 hours and 7 days.

RESULTS

    ______________________________________                                        Retention of Selenium-75 Radioactivity at                                                0        48 hrs.    7 days                                         ______________________________________                                        Normal (1)   100%       80%        31%                                        Normal (2)   100%       100%       27%                                        Ileal resection (3)                                                                        100%       26%        7.5%                                       Ileal resection (4)                                                                        100%       80%        9.5%                                       ______________________________________                                    

Patient (4) was an old inactive female who was on drugs to slow downbowel transfer.

The difference in retained radioactivity between normal patients (1) and(2) and ileal resection patients (3) and (4) is large enough to bereadily observed. The method offers the following advantages over theuse of carbon-14 labelled bile sal salts:

(a) Bowel function can be investigated by body counting of γ-radiation,without the need for faecal counting or for preparation of samples forcounting β-radiation.

(b) Because only small amounts of labelled bile salts are used, andbecause these are rapidly eliminated, the patient is subjected to onlylow levels of radiation.

The labelled compounds might conveniently have been administered in theform of a capsule containing the labelled bile salt adsorbed on acarrier.

The following preparative Examples further illustrate the invention.

EXAMPLE 1 The Preparation of a mixture of 19-methyl-⁷⁵ Seseleno-labelled bile salts

A male rabbit (NZW×LOP; 4.8 kg) was anaesthetized with sodiumpentobarbitone ("Sagatal"), intravenously injected. A tracheotomy wasperformed and into a jugular vein was inserted a cannula with a 3-waytap. The animal was ventilated by intermittent positive pressure andanaesthesia maintained by intravenous administration of pentobarbitoneas required. A midline ventral incision was made in the abdominal walland the liver reflected to reveal the gall bladder, cystic duct and thecommon bile duct. After ligation of the cystic duct the common bile ductwas cannulated for the collection of bile.

After a period of stabilization 1 ml of a solution of 19-methyl-[⁷⁵Se]selenocholesterol (0.01 mg; 12 mCi) in polysorbate/normal saline wasinjected via the jugular cannula. Bile was collected as a series of15-minute samples in preweighed tubes. After collection each sample wasweighed and counted for ⁷⁵ Se radioactivity. The flow of bile, initiallyat 3.4 ml/15 minutes, declined to 1.5 ml/15 minutes after 61/2 hours.During this period 56.65 g of bile was collected containingapproximately 100 μCi of ⁷⁵ Se radioactivity (about 1 percent of theinjected dose).

The labelled bile was added to 1000 ml of absolute ethanol which wasvigorously stirred and brought momentarily to boiling. The ethanolicsolution, after cooling, was filtered and reduced in volume to 10 ml. Asmall precipitate at this stage was again removed by filtration, and thefiltrate was evaporated to dryness in vacuo. The residual green gum wasextracted with 40°-60° petroleum ether (4×5 ml) to remove lipidmaterial, and then dissolved in methanol (2×5 ml) and the solutionfiltered. Yield of ⁷⁵ Se bile salts, 60 μCi. TLC: Kieselgel 60 F₂₅₄ ;chloroform, methanol 5:1 major component (>90%) R_(f) 0.00 (Inactivemarkers of glycocholic acid, R_(f) 0.00; glycocheno-deoxycholic acid,R_(f) 0.06; cholic acid, R_(f) 0.14; deoxycholic acid, R_(f) 0.70.)

The methanolic solution, containing both natural bile salts and ⁷⁵ Selabelled bile salts, was reduced in volume and applied to six PLCplates. (Kieselgel 60 F₂₅₄, 2 mm). The plates were eluted withchloroform, methanol (5:1), autoradiographed, and the component at R_(f)0.00 removed from the plates and extracted into methanol. Yield, 26 μCi.On treatment of a sample of this purified 19-methyl-[⁷⁵ Se]selenolabelled bile salt with the enzyme cholylglycine hydrolase thechromatographic mobility on Merck Kieselgel 60 F₂₅₄ (chloroform,methanol 5:1) changed from R_(f) 0.00 to R_(f) s 0.30 and 0.47).

EXAMPLE 2 The Preparation of 3α,12α-dihydroxy-22-(carboxymethyl-[⁷⁵Se]seleno)-23,24-bisnor-5β-cholane (23-Selena-25-homodeoxycholic Acid)

(i) 3α,12α-Diacetory-22-Iodo-23,24-bisnor-5β-cholane

3α,12α-Diacetoxy-24-nor-5β-cholanic acid (0.3 g) in dry carbontetrachloride (30 ml) was treated with dry, powdered, lead tetraacetate(0.3 g) and was heated to reflux in an atmosphere of dry nitrogen. Thesolution was irradiated with an Atlas 275 watt infra-red lamp and asolution of iodine (0.16 g) in dry carbon tetrachloride (12 ml) wasadded portionwise over a period of 10 minutes. The reaction mixture wasirradiated and stirred for a further 1 hour and was allowed to cool. Thesolution was filtered, the filtrate was washed successively with 5%sodium thiosulphate solution and water, and then dried over anhydroussodium sulphate. Evaporation of the solvent and crystallisation of theresidue from ethanol gave3α,12α-diacetoxy-22-iodo-23,24-bisnor-5β-cholane (0.3 g, 85%) m.p.172°-174°.

TLC (Merck Kieselgel 60 F₂₅₄ ; chloroform): Single component Rf0.50.

IR Spectrum: ν max: 2960, 2930, 2870, 1735, 1453, 1374, 1239, 1194, 1018cm⁻¹.

NMR (220 MH_(z), CDCl₃): τ 4.95 (1H,S,C₁₂ -proton); τ 5.32 (1H,M,C₃-proton); τ 6.76 (2H,M,C₂₂ --H), τ 7.86 (3H,S,12-Acetate protons), τ7.98 (3H,S,3-acetate protons), τ 8.00-9.05 (22H, steroid nucleus), τ9.10 (6H,S (with minor splitting), C₁₉ --H+C₂₁ --H), τ 9.23 (3H,S,C₁₈--H).

(ii) 23-Selena-25-homodeoxycholic acid-⁷⁵ Se

Red selenium-⁷⁵ Se was precipitated by bubbling sulphur dioxide througha solution of sodium selenite (15.9 mg) in water (2 ml) and concentratedhydrochloric acid (4 ml) containing sodium selenite-⁷⁵ Se (11.7 mCi, 1.2mg selenium). The precipitate was centrifuged off, it was washedthoroughly with de-ionised water and dried over phosphorus pentoxideunder vacuum.

Red selenium-⁷⁵ Se (8.4 mg, 0.11 mA, 109 mCi/mA) was suspended inethanol (2 ml) and potassium cyanide (7 mg, 0.11 mmole) was added; themixture was stirred at room temperature for two hours until completesolution had occurred. Redistilled ethyl bromoacetate (12 μl) was addedto the solution at 0° C. and it was stirred for 11/2 hours.3α,12α-Diacetoxy-22-iodo-23,24-bisnor-5β-cholane (60 mg) in drytetrahydrofuran (1 ml) was added to sodium borohydride (9 mg) in ethanol(1 ml). The reaction mixture was cooled in ice and the ethanolicsolution of ethyl selenocyanatoacetate-⁷⁵ Se was added over a period of10 minutes. Stirring was continued for a further 2 hours while thetemperature rose to room temperature. Acetone (1 ml) was added and thesolution was evaporated under reduced pressure. Chloroform (2 ml) wasadded to the residue, insoluble material was removed by filtration andthe solution was concentrated to a small bulk. The required product wasisolated by preparative layer chromatography (Anachem Silica Gel GF, 1mm; chloroform, methanol 20:1). The major component, Rf0.85, as observedby autoradiography, was removed from the plate and extracted into ethylacetate (3×4 ml). Yield of ethyl3α,12α-diacetoxy-23-selena-25-homo-5β-cholanate-⁷⁵ Se, 6.1 mCi.

IR Spectrum: ν max: 2935, 2860, 1735, 1450, 1378, 1245, 1050, 750 cm⁻¹.

The solution was evaporated and sodium hydroxide (100 mg) in ethanol (5ml) and water (1 ml) was added. The solution was stirred and heatedunder reflux for 2 hours; it was then cooled and evaporated. Water (3ml) was added, the solution was filtered from some insoluble materialand acidified by the addition of Bio-Rad AG 50W-X12 cation exchangeresin in the H+ form. The resin was removed by filtration, it was washedwith methanol (3 ml) and the combined filtrate was evaporated. Theresidue was dissolved in the minimum of methanol and the product wasisolated by preparative layer chromatography (Anachem Silica Gel GF, 1mm; chloroform, methanol 6:1). The required band, Rf0.42, was located byautoradiography; it was removed from the plate and isolated byextraction into methanol. Evaporation of the solvent afforded23-selena-25-homodeoxycholic acid-⁷⁵ Se(2.4 mCi).

TLC (Merck Kieselgel 60 F₂₅₄):

(a) Chloroform, methanol-5:1; Major component (95%) Rf0.36

(b) Iso octane, diisopropyl ether, acetic acid-2:1:1; Major componentRf0.43

IR Spectrum: ν max: 3380, 2930, 2860, 1700, 1448, 1380, 1255, 1105, 1035cm⁻¹.

(iii) Tauro-23-selena-25-homodeoxycholic acid-⁷⁵ Se

23-Selena-25-homodeoxycholic acid-⁷⁵ Se (0.27 mCi, 2.0 mg) was treatedwith a solution of N-ethoxycarbonyl-2-ethoxy-dihydroquinoline (3 mg) indry dimethylformamide (620 μl) and stirred for 30 minutes. The solutionwas added to a mixture of taurine (1.55 mg) in dimethylformamide (350μl) containing triethylamine (3.3 μl) and the reaction mixture washeated at ca. 90° for 30 minutes. After standing at ambient temperatureovernight, water (1 ml) was added, the solution was acidified byaddition of concentrated hydrochloric acid and evaporated. Ethanol (0.5ml) was added to the residue and the product was isolated by preparativelayer chromatography (Anachem Silica Gel GF, 1 mm; chloroform,methanol-5:2). The product band, Rf0.32, was removed from the plate andthe product was isolated by extraction with methanol. Evaporation of thesolvent gave tauro-23-selena-25-homodeoxycholic acid-⁷⁵ Se (0.14 mCi).

TLC (Merck Kieselgel 60 F₂₅₄ ; chloroform, methanol 3:1): MajorComponent (94%) Rf0.34 (cf 23-selena-25-homodeoxycholic acid-Rf 0.65 inthe same system).

IR Spectrum: ν max: 3400, 2940, 2870, 1698, 1650, 1545, 1390, 1208,1180, 1070 cm⁻¹.

(iv) Ethyl 3α,12α-Diacetoxy-23-Selena-25-homo-5β-cholanate and23-Selena-25-homodeoxycholic Acid

Non-radioactive ethyl 3α,12α-diacetoxy-23-selena-25-homo-5β-cholanateand 23-selena-25-homodeoxycholic acid were prepared by the methoddescribed in 2(ii). Quantities of reagents used:- ethylselenocyanatoacetate, 35 mg in 0.7 ml ethanol; sodium borohydride, 12.6mg; 3α,12α-diacetoxy-23-iodo-23,24-bisnor-5β-cholane, 100 mg; ethanol, 5ml; tetrahydrofuran, 1 ml. Yield of ethyl3α,12α-diacetoxy-23-selena-25-homo-5β-cholanate 64 mg.

IR Spectrum: ν max: 2940, 2865, 1738, 1450, 1380, 1245, 1105, 1060,cm⁻¹.

NMR (220 MHz, CDCl₃): τ 4.93 (1H,S,C₁₂ -proton), τ 5.32 (1H,M,C₃-proton), τ 5.84 (2H,q,ethyl CH₂), τ 6.90 (2H,S,C₂₄ -protons), τ 7.06(1H,M,C₂₂ -proton), τ 7.45 (1H,q,C₂₂ -proton), τ 7.90 (3H,S,12-acetateprotons), τ 7.96 (3H,S,3-acetate protons), τ 8.72 (3H,t,ethyl CH₃), τ9.08 (3H,d,C₂₁ -protons), τ 9.12 (3H,S,C₁₉ -protons), τ 9.25 (3H,S,C₁₈-protons), τ 8.0-9.25 (22H, steroid nucleus).

Ethyl 3α,12α-diacetoxy-23-selena-25-homo-5β-cholanate (120 mg) wasdissolved in ethanol (5 ml) and hydrolysed as described in 2 (ii) giving23-selena-25-homodeoxycholic acid (45 mg).

TLC (Merck Kieselgel 60 F₂₅₄ ; chloroform, methanol 5:1): The product,visualised by exposure to iodine vapour, chromatographed as a singlecomponent (Rf 0.32) and coincided with the radioactive marker.

IR Spectrum: ν max: 3430, 2920, 2855, 1700, 1448, 1375, 1255, 1038 cm⁻¹.

NMR (220 MHz, CD₃ OD): τ 5.12 (solvent peak), τ 6.05 (1H,S,C₁₂ -proton),τ 6.50 (1H,m,C₃ -proton), τ 6.7 (solvent peak), τ 6.93 (2H,S,C₂₄-protons), τ 7.07 (1H,m,C₂₂ -proton), τ 7.54 (1H,q,C₂₂ -proton), τ 7.85(3H,S,CH₃ CO₂ H), τ 8.88 (3H,d,C₂₁ -protons), τ 9.07 (3H,S,C₁₉-protons), τ 9.28 (3H,S,C₁₈ -protons), τ 8.0-9.2 (22H,steroid nucleus).

(v) 23-Selena-25-homodeoxycholic acid selenoxide-⁷⁵ Se

23-Selena-25-homodeoxycholic acid-⁷⁵ Se [68.4 μCi, 1.1 μmole] inmethanol (1.0 ml) was treated with an aqueous solution of hydrogenperoxide (5 μl, 4 μmole) and was allowed to stand at ambient temperaturefor 90 minutes.

TLC (Merck Kieselgel 60 F₂₅₄, dichloromethane, acetone, aceticacid-7/2/1): Major Component (greater than 90%) Rf 0.19 (cf23-Selena-25-homodeoxycholic acid-⁷⁵ Se Rf 0.84 in this system).

EXAMPLE 3 Preparation of3α,7α-Dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane

(i)3α,7α-Dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane-3,7-dinitrate-⁷⁵Se

Red selenium-⁷⁵ Se (5.0 mg, 6.4 mCi) was prepared as described inExample 2 (ii) and was suspended in de-ionised water (0.55 ml).Potassium cyanide (4 mg) was added and the mixture was stirred until allthe selenium had dissolved. β-Propiolactone (5 μl) was added and afterstirring for 15 minutes the solution was acidified by the dropwiseaddition of concentrated hydrochloric acid (some red selenium wasprecipitated) and evaporated. Ether (3 ml) was added to the residue andthe solution of β-selenocyanatopropionic acid-⁷⁵ Se was filtered toremove insoluble products and evaporated (5.4 mCi).

3α,7α-Dihdroxy-23-bromo-24-nor-5β-cholane-3,7-dinitrate (30.8 mg) wasdissolved in tetrahydrofuran (1.0 ml) and was added to sodiumborohydride (8.3 mg) in ethanol (0.7 ml). The solution was cooled in iceand β-selenocyanatopropionic acid-⁷⁵ Se in ethanol (1.0 ml) was added inportions over 10 minutes. After a further 1 hour, acetone (1 ml) wasadded, the solution was acidified with concentrated hydrochloric acidand evaporated to dryness. The residue was extracted into ether and thesolution was filtered from insoluble material. TLC (Merck Kieselgel 60F₂₅₄ ; chloroform, methanol 10:1) demonstrated three major radioactiveproducts Rf 0.97, 0.85 and 0.09. Component Rf 0.85 corresponded toinactive marker (Example 3 (iii)).

The product was isolated by preparative layer chromatography (AnachemSilica Gel GF, 1 mm; chloroform; methanol - 10:1). It was located byautoradiography (Rf 0.41), removed from the plate and extracted intoether (3×3 ml) giving 1.1 mCi of3α,7α-dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane-3,7-dinitrate.

TLC (Merck Kieselgel 60 F₂₅₄ ; chloroform, methanol 10:1): Majorcomponent (95%) Rf 0.54 corresponds to non-radioactive standard.

(ii) 3α,7α-Dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane-⁷⁵ Se

The dinitrate (1.1 mCi - prepared as described above - 3 (i)) wasdissolved in glacial acetic acid (1 ml) and zinc dust (60 mg) was addedin portions. The reaction mixture was stirred at ambient temperature for1 hour and stored at -20° C. overnight. After warming to roomtemperature the solution was filtered and the filtrate was lyophilized.The product was isolated by preparative layer chromatography (AnachemSilica Gel GF, 1 mm; chloroform, methanol (7:1). It was located byautoradiography (Rf 0.30), removed from the plate and extracted intomethanol to give3α,7α-dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane (0.6 mCi.

TLC (Merck Kieselgel 60 F₂₅₄):

(a) chloroform methanol, 5:1; major component (97%) Rf 0.65

(b) chloroform, methanol; 10:1; major component Rf 0.22

(c) isooctane, diisopropylether, acetic acid; 2:1:1; major component Rf0.41

In each case the product coincided with the non-radioactive standard.

(iii)3α,7α-Dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane-3,7-dinitrate

Non-radioactive3α,7α-dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane was preparedby the method described (3(i)) using the quantities of reagents asfollows: 3α,7α-dihydroxy-23-bromo-24-nor-5β-cholane-3,7-dinitrate (173.1mg) in tetrahydrofuran (4 ml); sodium borohydride (45.8 mg) in ethanol(2.2 ml) and β-selenocyanatopropionic acid (61.4 mg) in ethanol (2.2ml). The reaction mixture was treated with acetone (1 ml), it was pouredinto water (25 ml), acidified with concentrated hydrochloric acid andextracted with ether (2×20 ml). The combined ether extracts were washedwith 5% sodium carbonate solution (2×20 ml) and the combined alkalineextracts were acidified. The precipitate was isolated by ether, theextracts were dried and evaporated. The product was purified bypreparative layer chromatography (Merck Kieselgel F₂₅₄, 2mm--chloroform, methanol (10:1). The required band was located underu.v., it was removed from the plate and extracted into ether.Evaporation of the solvents left 3α,7α-dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane-3,7-dinitrate as a whitesolid (82 mg).

IR Spectrum: ν max: 3450, 2940, 1710, 1620, 1278, 862 cm⁻¹.

NMR (220 MHz, CDCl₃): τ 4.95 (1H, S C₇ -proton), τ 5.22 (1H, m, C₃proton), τ 7.23 (4H, S, C₂₅ and C₂₆ -protons), τ 7.6 (2H, m, C₂₃-protons), τ 9.05 (6H, s+d, C₁₉ -protons and C₂₁ -protons), τ 9.32 (3H,S, C₁₈ -protons), τ 7.85-9.10 (24H, steroid nucleus).

(iv) 3α,7α-Dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane

3α,7α-Dihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane (50 mg) wasprepared from its dinitrate ester (80 mg) by the method described(3(ii)).

IR Spectrum: ν max: 3435, 2940, 2870, 1715, 1550, 1410, 1300, 1080, 960cm⁻¹.

NMR (220 MHz, CD₃ OD): τ 5.16 (solvent peak), τ 6.20 (1H, S, C₇-proton), τ 6.94 (1H, m, C₃ -proton), τ 6.99 (solvent peak), τ 7.25 (4H,S, C₂₅ and C₂₆ -protons), τ 7.45 (2H, m, C₂₃ protons), τ 9.02 (3H, d,C₂₁ -protons), τ 9.07 (3H, S, C₁₉ -protons), τ 9.29 (3H, S, C₁₈-protons).

EXAMPLE 4 Preparation of3α,7α,12α-Trihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane

(i) Cholic Acid Triformate

Cholic acid (50 g) was treated with 100% formic acid (240 ml) and thewhole was stirred at 70°-80° C. for 6 hours. The solution was cooled andmost of the solvent was evaporated. The residue was triturated withether (500 ml) giving a white solid which was filtered and dried (43 g).The crude product could be further purified by successiverecrystallisation from 60% aqueous ethanol and 1:1 60°-80° petrol,acetone. M.p. of purified material 204°-208° C.

(ii) 3α,7α,12α-Triformoxy-23-Iodo-24-nor-5βcholane

Cholic acid triformate (1.06 g) and lead tetracetate (0.97 g) weresuspended in dry carbon tetrachloride (100 ml) and the suspension wasstirred and heated to reflux in an atmosphere of dry nitrogen. Refluxwas maintained by irradiation with an Atlas 275 watt infra-red lamp anda solution of iodine (0.52 g) in carbon tetrachloride (40 ml) was addedin portions. Reflux was continued for a further 1 hour. The reactionmixture was allowed to cool and then filtered.

The filtrate was washed successively with 5% sodium thiosulphatesolution and water, and was dried over anhydrous sodium sulphate.Evaporation of the solvent and recrystallisation of the residue fromethanol (twice) gave 3α,7α,12α-triformoxy-23-iodo-24-nor-5β-cholane(0.65 g) as colourless crystals, m.p. 166°-168°.

IR Spectrum: ν max: 2960, 2938, 2862, 2712, 1721, 1518, 1360, 1160,1060, 995, 600 cm⁻¹.

NMR (220 MHz, CDCl₃): τ 1.85, 1.90, 1.98 (3H, 3 singlets, 3-7-and12-formate protons), τ 4.74 (1H, S, C₁₂ -proton), τ 4.94 (1H, S, C₇-protons), τ 5.30 (1H, m, C₃ -proton), τ 6.72+6.95 (2H, m, C₂₃ protons),τ 9.06 (3H, S, C₁₉ -protons), τ 9.15 (3H, d, C₂₁ -protons), τ 9.22 (3H,S, C₁₈ -protons), τ 7.8-9.05 (22H, steroid nucleus).

(iii)3α,7α,12α-Trihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane-⁷⁵ Se

β-Selenocyanatopropionic acid-⁷⁵ Se (4.42 mCi. 108 mCi/mmole) wasprepared from red selenium-⁷⁵ Se as described for Example 3(i).3α,7α,12α-Triformoxy-23-iodo-24-nor-5β-cholane (23 mg) intetrahydrofuran (0.5 ml) was added to Sodium borohydride (5.5 mg) inethanol (0.5 ml) and the solution was cooled in ice.β-Selenocyanatopropionic acid-⁷⁵ Se (4.42 mCi) in ethanol (0.8 ml) wasadded to the solution over a period of 10 minutes and stirring wasallowed to continue for 1 hour. The reaction mixture was treated withacetone (1 ml), acidified with concentrated hydrochloric acid, andevaporated. The residue was partitioned between ether and water and theethereal phase was separated and extracted with 5% aqueous sodiumcarbonate solution. The alkaline extract was acidified and theprecipitate was isolated by ether extraction.

Ethanol (2 ml), water (0.75 ml) and potassium hydroxide (100 mg) wasadded to the crude sample of3α,7α,12α-triformoxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane. Thesolution was stirred at ambient temperature for 2 hours, it was thenacidified and evaporated. Methanol (2 ml) was added to the residue, thesolution was filtered from insoluble material and concentrated to smallbulk. The product was purified by preparative layer chromatography(Merck Kieselgel 60 F₂₅₄ 1 mm; chloroform, methanol 5:1). The requiredband was located by autoradiography (Rf 0.35); it was removed from theplate and extracted into methanol to give3α,7α,12α-trihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane-⁷⁵ Se(1.2 mCi).

TLC (Merck Kieselgel 60 F₂₅₄ :

(a) chloroform, methanol 5:1--major component (95%) Rf 0.57 correspondedto non-radioactive standard

(b) isooctane, diisopropylether, acetic acid 2:1:1; Rf 0.21

IR Spectrum: ν max: 3520, 3416, 2930, 2870, 1740, 1718, 1440, 1380,1322, 1170, 1080 cm⁻¹.

(iv) 3α,7α,12α-Trihydroxy-23-carboxyethylseleno)-24-nor-5.beta.-cholane

Non-radioactive3α,7α,12α-trihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane wasprepared by the method described in 4(iii). The following quantities ofreagents were used: 3α-7α,12α-triformoxy-23-iodo-24-nor-5β-cholane,258.7 mg; sodium borohydride, 61.2 mg; β-selenocyanatopropionic acid,79.6 mg. Following the final hydrolysis step the product was purified bypartition between ether and 5% sodium carbonate solution. The acidicproduct was isolated and triturated with acetone to give3α,7α,12α-trihydroxy-23-(β-carboxyethylseleno)-24-nor-5β-cholane (70 mg)as a white powder, m.p. 198°-200°.

IR Spectrum: ν max: 3520, 3410, 2930, 2870, 1740, 1718, 1440, 1382,1323, 1170, 1080 cm⁻¹.

NMR (220 MHz, CD₃ OD): τ 5.11 (solvent peak), τ 6.06 (1H, S, C₁₂-proton), τ 6.23 (1H, S, C₇ -proton), τ 6.67 (1H, m, C₃ -proton), τ 6.71(solvent peak), τ 7.30 (4H, S, C₂₅ +C₂₆ -protons), τ 7.47 and τ 7.78(2H, m, C₂₃ -protons), τ 8.97 (3H, d, C₂₁ -protons), τ 9.10 (3H, S, C₁₉-protons), τ 9.30 (3H, S, C₁₈ -protons), τ 9.70 (unidentified).

EXAMPLE 5 Preparation of3α,7α,12α-trihydroxy-20-(carboxy-methylseleno)-5β-pregnane(22-selenacholic acid)

(i) 3α,7α,12α-Triformoxy-Δ²² -24-nor-5β-cholene

Cupric Acetate dihydrate (1.0 g) and pyridine (0.7 ml) were added tobenzene (170 ml) and the suspension was dried by azeotropic distillationusing a Dean and Stark apparatus. After cooling somewhat, dry leadtetraacetate (20 g) and cholic acid triformate (10.5 g, prepared asdescribed in 4(i) were added and the reaction mixture was stirred andheated under reflux in an atmosphere of dry nitrogen for 11/2 hours. Itwas allowed to cool and was filtered. The filtrate was washedsuccessively with water, 1 M sodium hydroxide solution and finally withwater, and was dried over anhydrous sodium sulphate. Evaporation of thesolvent and crystallisation of the residue from ethanol gave3α,7α,12α-triformoxy-Δ²² -24-nor-5β-cholene (4.0 g) m.p. 188°-190°.

IR Spectrum: ν max: 3077, 2960, 2865, 1725, 1714, 1637, 1468, 1449,1380, 1180 cm⁻¹.

NMR Spectrum: τ 1.83, 1.91, 1.98 (3H, three singlets, 3-, 7- and 12-formate protons), τ 4.4 (1H, m, C₂₂ -proton), τ 4.77 (1H, S, C₁₂-proton), τ 4.97 ) 1H, S, C₇ -proton), τ 5.16 (1H, d, C₂₃ -proton(cis)), τ 5.18 (1H, S, C₂₃ -proton (trans), τ 5.30 (1H, m, C₃ -proton),τ 9.07 (6H, s+d, C₁₉ -protons+C₂₁ -protons), τ 9.24 (3H, S, C₁₈-protons), τ 7.75-τ 9.1 (22H, steroid nucleus).

(ii) 3α,7α,12α-Triformoxy-23,24-bisnor-5β-cholanic acid

3α,7α,12α-Triformoxy-Δ²² -24-nor-5β-cholene (2.4 g) was dissolved in2-methylpropan-2-ol (800 ml) and potassium carbonate (1.41 g) in water(800 ml) was added. Sodium periodate (20.86 g) and potassiumpermanganate (0.395 g) were dissolved in water (1 liter) and an aliquot(435 ml) was added to the solution of the olefin. The solution wasstirred at ambient temperature for 24 hours. Sufficient 40% sodiumhydrogen sulphite solution was added to discharge the permanganatecolouration, 5% sodium carbonate solution was added to pH 8, and thesolution was concentrated at reduced pressure to ca. 250 ml. It wasextracted with chloroform (2×100 ml), treated with further 40% sodiumhydrogen sulphite and acidified with concentrated hydrochloric acid. Themixture was extracted with chloroform (4×100 ml), and the combinedextracts were washed successively with 5% sodium thiosulphate solutionand water, and then dried. The solvent was evaporated and 100% formicacid (30 ml) was added to the residue. The solution was stirred andheated at 70°-80° for 6 hours and was allowed to cool. It was pouredinto water and the precipitate was extracted into chloroform (3×50 ml).The combined organic extracts were washed with water, dried andevaporated. The residue was recrystallised from ethanol to give3α,7α,12α-triformoxy-23,24-bisnor-5β-cholanic acid (0.8 g) m.p.165°-170°.

IR Spectrum: ν max: 3410, 2965, 2940, 2870, 1722, 1450, 1385, 1178, 890cm⁻¹.

NMR Spectrum (220 MHz, CDCl₃): τ 1.83, 1.91 and 1.98 (3H, 3 singlets,3-, 7- and 12-formate protons), τ 4.78 (1H,S,C₁₂ -proton), τ 4.93(1H,S,C₇ -proton), τ 5.30 (1H,m,C₃ -proton), τ 6.29 (2H,q,CH₂ of ethanolof crystallisation), τ 7.64 (1H,q,C₂₀ -proton), τ 8.77 (3H,t,CH₃ ofethanol of crystallisation), τ 8.88 (3H,d,C₂₁ -protons), τ 9.05 (3,S,C₁₉-protons), τ 9.22 (3H,S,C₁₈ -protons), τ 7.75-9.05 (19H, steroidnucleus).

(iii) 3α,7α,12α-Triformoxy-20-iodopregnane

3α,7α,12α-Triformoxy-23,24-bisnor-5β-cholanic acid (0.2 g) was convertedto 3α,7α,12α-Triformoxy-20-iodopregnane (0.11 g) by the method describedin 2(i) m.p. 145°-146.5° (decomp).

IR Spectrum: ν max: 3405, 2950, 2860, 1713, 1445, 1377, 1180 cm⁻¹.

NMR Spectrum (220 MHz, CDCl₃): τ 1.81, 1.91 and 1.98 (3H, 3 singlets,3-, 7- and 12-formate protons), τ 4.75 (1H,S,C₁₂ -proton), τ 4.93(1H,S,C₇ -proton), τ 5.30 (1H,m,C₃ -proton), τ 5.80 (1H,q,C₂₀ -proton),τ 8.06 (3H,d,C₂₁ -protons), τ 9.07 (3H,S,C₁₉ -protons), τ 9.25 (3H,S,C₁₈-protons), τ 7.5-9.0 (19H, steroid nucleus).

(iv) 22-Selenacholic Acid-⁷⁵ Se

Red selenium-⁷⁵ Se (8.2 mg, 106 mCi/mA) was prepared as described in2(ii). It was suspended in ethanol (2 ml) and dry nitrogen was bubbledthrough the solution. The exit gases were passed through a trapcontaining 5% lead acetate solution. Sodium borohydride (2.7 mg) wasadded and the suspension was stirred at ambient temperature for 20minutes. n-Propanol (5 ml) was added and the reaction mixture was heatedon a boiling water bath for 20 minutes.3α,7α,12α-Triformoxy-20-iodopregnane (35 mg) in warm n-propanol (2 ml)was added to the solution of disodium diselenide-⁷⁵ Se and the whole washeated on a boiling water bath in an atmosphere of dry nitrogen for 31/2hours. It was allowed to cool; it was evaporated under reduced pressureand the residue was treated with chloroform (5 ml). The solution wasfiltered and evaporated to dryness leaving the impure dipregnanediselenide-⁷⁵ Se (4.2 mCi).

Sodium borohydride (5 mg) was dissolved in ethanol (1 ml), the solutionwas cooled in ice and ethyl bromoacetate (20 μl) was added. Thedipregnane diselenide-⁷⁵ Se was dissolved in ethanol (3 ml) and wasadded dropwise over a period of 10 minutes. The reaction mixture wasstirred for 2 hours, acetone (1 ml) was added and the solution wasevaporated. Chloroform (3 ml) was added, inorganic salts were removed byfiltration, and the solution was treated with sodium hydroxide (100 mg)in water (1 ml). The solution was heated under reflux for 3 hours,cooled and evaporated. The residue was dissolved in water (3 ml) and thesolution was acidified with concentrated hydrochloric acid andlyophilized. Acetic acid (3 ml) was added to the residue, the solutionwas filtered and concentrated to a small bulk. The product was purifiedby preparative layer chromatography, (Anachem Silica Gel GF, 1 mm;dichloromethane, acetone, acetic acid, 7:2:1). Its location wasdetermined by autoradiography, the band was removed from the plate andthe product was extracted into acetic acid and the solvent evaporated togive 22-selenacholic acid-⁷⁵ Se (0.8 mCi).

TLC (Merck Kiesgel 60 F₂₅₄):

(a) Dichloromethane, acetone, acetic acid; (7:2:1)

Major component Rf 0.22

(b) Chloroform, methanol; (5:1)

Major component Rf 0.11

IR Spectrum: ν max: 3400, 2925, 2780, 1715, 1440, 1375, 1265, 1073, 1040cm⁻¹.

(v) Glyco-22-selenacholic acid-⁷⁵ Se

22-Selenacholic-⁷⁵ Se (0.40 mCi; 1.9 mg) in acetic acid was evaporatedto dryness. Dry ethyl acetate (450 μl) was added followed byN-ethoxycarbonyl-2-ethoxydihydroquinoline (14.2 mg). Ethyl glycinatehydrochloride (8.0 mg), suspended in dry ethyl acetate (0.6 ml), wastreated with triethylamine (8.3 μl); the mixture was stirred for 30minutes and was added to the solution of 22-selenacholic acid-⁷⁵ Se, afurther quantity of ethyl acetate (0.4 ml) was used to complete thetransfer. The reaction mixture was heated under reflux on a boilingwater bath for 6 hours; it was then cooled and evaporated. Chloroform (4ml) was added to the residue and insoluble material was removed byfiltration.

Ethyl 22-selenaglycocholate-⁷⁵ Se was purified by preparative layerchromatography (Anachem Silica Gel GF, 1 mm; chloroform, methanol 8:1).The major radioactive band was located by autoradiography, Rf 0.4; itwas removed from the plate and extracted into methanol (3×4 ml). Thesolvent was evaporated, ethanol (4 ml) and 10% potassium carbonatesolution (1 ml) were added and the solution was heated under reflux for1 hour and allowed to stand at room temperature overnight. The solutionwas acidified with concentrated hydrochloric acid, evaporated to drynessand the product was extracted from the residue by dissolving in ethanol.The solution was filtered and evaporated leaving glyco-22-selenacholicacid-⁷⁵ Se (0.21 mCi).

TLC (Merck Kieselgel 60 F₂₅₄ ; chloroform, methanol 3:1): Majorcomponent (ca. 85%) Rf 0.04 (cf 22-Selenacholic acid, Rf 0.31 andglycocholic acid, Rf 0.02, in this system).

EXAMPLE 6 Preparation of 3α-Hydroxy-24-(carboxymethylseleno)-5β-cholane

(i) 3α-Acetoxy-25-homo-5β-cholanic acid

3α-Acetoxy-25-homo-5β-cholanic acid was prepared from lithocholic acidusing the Arndt-Eistert reaction for lengthening the C₁₇ side chain.

(ii) 3α-Acetoxy-24-iodo-5β-cholane

3α-Acetoxy-25-homo-5β-cholanic acid was transformed to3α-Acetoxy-24-iodo-5β-cholane by the method quoted in 4(ii). Thequantities of reagents used were as follows:3α-acetoxy-25-homo-5β-cholanic acid (1.8 g) in dry carbon tetrachloride(120 ml), lead tetraacetate (2.0 g) and iodine (1.04 g) in carbontetrachloride (80 ml). The crude product was purified by preparativelayer chromatography using five Merck Kieselgel 60 F₂₅₄, 2 mm platesdeveloped in chloroform. The required uv. absorbing band was removedfrom each plate and the product was isolated by extraction with ether.Evaporation of the solvent and trituration of the residue with ethanolgave 3α-acetoxy-24-iodo-5β-cholane (0.43 g; m.p. 140°-146°) as a whitepowder.

IR Spectrum: ν max: 2940, 2865, 1738, 1473, 1459, 1383, 1366, 1258, 1028cm⁻¹.

NMR Spectrum (220 MHz, CDCl₃): τ 5.19 (1H, m,C₃ -proton), τ 6.83(2H,m,C₂₄ -protons), τ 7.98 (3H,S,acetate protons), τ 9.07 (6H,1s+1d,C₁₉ +C₂₁ -protons), τ 9.36 (3H,S,C₁₈ -protons), τ 8.0-9.1 (28H, steroidnucleus).

(iii) 3α-Hydroxy-24-(carboxymethylseleno)-5β-cholane-⁷⁵ Se

Ethyl selenocyanatoacetate-⁷⁵ Se (17 mg, 9.2 mCi) was prepared in themanner previously described (2 (ii) ). It was reacted with sodiumborohydride (8.2 mg) in ethanol (2 ml) and 3α-acetoxy-24-iodo-5β-cholane(50 mg) in tetrahydrofuran (3 ml) as described in 2(ii). Theintermediate 3α-acetoxy-24-(carboxymethylseleno)-5β-cholane ethylester-⁷⁵ Se was isolated by preparative layer chromatography (AnachemSilica Gel GF; chloroform). The main radioactive band was located byautoradiography (Rf 0.55); it was removed from the plate and the productwas isolated by extraction with ethylacetate (3×4 ml). The solvent wasevaporated, ethanol (5 ml) and potassium hydroxide (100 mg) in water (1ml) were added and the solution was heated under reflux for 3 hours andallowed to cool. The solution was acidified with concentratedhydrochloric acid and evaporated under reduced pressure. Ethanol (1 ml)was added to the residue, the solution was filtered and the productisolated by preparative layer chromatography (Anachem Silica Gel GF;chloroform, methanol; 12:1). The required band (Rf 0.20) was located byautoradiography, it was removed from the plate and the product wasisolated by extraction with ethanol. Evaporation of the solvent gave3α-hydroxy-24-(carboxymethylseleno)-5β-cholane-⁷⁵ Se (0.8 mCi).

TLC (Merck Kieselgel 60 F₂₅₄ ; dichloromethane, methanol-15:1): MajorComponent (94%) - Rf 0.25, coincided with the non-radioactive standard.

IR Spectrum: ν max: 3400, 2930, 2855, 1700, 1445, 1373, 1105, 1028 cm⁻¹.

(iv) 3α-Acetoxy-24-(carboxymethylseleno)-5β-cholane ethyl ester

Non-radioactive 3α-acetoxy-24-(carboxymethylseleno)-5β-cholane ethylester (160 mg) was prepared by the method given in 6 (iii) from3α-acetoxy-24-iodo-5β-cholane (200 mg), sodium borohydride (32 mg) andethyl selenocyanatoacetate (74.7 mg).

IR Spectrum: ν max: 2925, 2855, 1733, 1445, 1375, 1360, 1238, 1100, 1023cm⁻¹.

NMR Spectrum (220 MHz, CDCl₃): τ 5.29 (1H,m,C₃ -proton), τ 5.83(2H,q,ethyl CH₃), τ 6.86 (2H,S,C₂₆ -protons), τ 7.98 (3H,S,acetateprotons), τ 8.72 (3H,q,ethyl CH₃), τ 9.0 (6H,1s+1d, C₁₉ -protons+C₂₁-protons), τ 9.36 (3H,S,C₁₈ -protons).

(v) 3α-Hydroxy-24-(carboxymethylseleno)-5β-cholane

Hydrolysis of 3α-acetoxy-24-(carboxymethylseleno)-5β-cholane ethyl esteraccording to the method in 6 (iii) gave3α-hydroxy-24-(carboxymethylseleno)-5β-cholane m.p. 117°-121° C.

IR Spectrum: ν max: 3440, 2920, 2855, 1705, 1443, 1372, 1270, 1165,1105, 1026 cm⁻¹.

EXAMPLE 7 Preparation of23-(Carboxymethylseleno)-24-nor-5β-cholane-3,7,12-trione-⁷⁵ Se

(i) 23-Iodo-24-nor-5β-cholane-3,7,12-trione

5β-Cholanic acid-3,7,12-trione was converted to23-iodo-24-nor-5β-cholane-3,7,12-trione by the method described in 4(ii). The quantities of reagents used were as follows: 5β-cholanicacid-3,7,12-trione (2 g) in carbon tetrachloride (200 ml), leadtetraacetate (2.3 g), iodine (1.2 g) in carbon tetrachloride (100 ml).The product was recrystallised successively from ethanol and petrol(60°-80°)-ethyl acetate, m.p. 256°-257° C.

TLC (Merck Kieselgel 60 F₂₅₄, chloroform): Major Component Rf 0.36 (if5β-cholanic acid-3,7,12-trione, Rf 0.08 in this system).

IR Spectrum: ν max: 2960, 2930, 1727, 1708, 1472, 1438, 1392, 1382,1304, 1280, 1226 cm⁻¹.

(ii) 23-(Carboxymethylseleno)-24-nor-5β-cholane-3,7,12-trione-⁷⁵ Se

An ethanolic solution of ethyl selenocyanatoacetate-⁷⁵ Se (15.3 mg, 8.8mCi) was prepared by the method described in 2 (ii); it was added to asolution of sodium borohydride (6.6 mg) in ethanol (1 ml) at 0°. Afterstirring at 0° for 20 minutes, acetone (1 ml) was added followed by23-Iodo-24-nor-5β-cholane-3,7,12-trione (39 mg) in tetrahydrofuran (1ml). The reaction mixture was stirred at ambient temperature for 16hours. The solution was evaporated, chloroform (2 ml) was added and,after filtration, the solution was concentrated and applied to anAnachem 1 mm silica plate which was developed in chloroform, methanol20:1. Three main radioactive bands were located by autoradiography (Rfs0.33, 0.49, 0.69); they were removed separately from the plate and theradioactive component was isolated from each by extraction with ether,ethanol (10:1). An examination of the separated components by thin layerchromatography (Merck Kieselgel 60 F254; chloroform) and by infra-redspectroscopy indicated that component Rf 0.49 was the required23-(carboxymethylseleno)-24-nor-5β-cholane-3,7,12-trione ethyl ester-⁷⁵Se, component Rf 0.33 was a mixture of two unidentified compounds andcomponent Rf 0.69 was non-steroidal.

23-(Carboxymethylseleno)-24-nor-5β-cholane-3,7,12-trione ethyl ester-⁷⁵Se (2.05 mCi) was dissolved in ethanol (5 ml) and 10% potassiumcarbonate solution (1 ml) was added. The solution was heated underreflux for 2 hours, cooled and evaporated under reduced pressure. Water(4 ml) was added, some insoluble material was removed by filtration andthe solution was acidified with concentrated hydrochloric acid andlyophilized. Chloroform (0.5 ml) was added to the residue and theproduct was isolated by preparative layer chromatography (Anachem SilicaGel Gf, 1 mm; chloroform, methanol-10:1). The main radioactive band waslocated by autoradiography (Rf 0.32); it was removed from the plate andthe product was isolated by extraction with methanol. Evaporation of thesolvent gave 23-(carboxymethylseleno)-24-nor-5β-cholane-3,7,12-trione-⁷⁵Se (1.3 mCi).

TLC (Merck Kieselgel 60 F₂₅₄ ; chloroform, methanol 20:1): Majorcomponent--greater than 95% Rf 0.31.

IR Spectrum: ν max: 2965, 2895, 1717, 1475, 1428, 1395, 1275, 1118 cm⁻¹.

EXAMPLE 8 Preparation of3α,12α-dihydroxy-23-(carboxymethyltelluro)-24-nor-5β-cholane

(i) 3α,12α-Diformoxy-23-Iodo-24-nor-5β-cholane

3α,12α-Diformoxy-5β-cholanic acid was prepared from deoxycholic acid (25g) and 100% formic acid ) 100 ml) by the method described in 4 (i). Theproduct was recrystallised from ethanol giving colourless crystals (17.5g), m.p. 197°-199° C.

3α,12α-Diformoxy-5β-cholanic acid (4 g) was converted to3α,12α-Diformoxy-23-iodo-24-nor-5β-cholane by the method previouslydescribed (4 (ii)) using lead tetraacetate (4.0 g) and iodine (1.9 g).The crude product was crystallised from ethanol giving colourlesscrystals m.p. 123°-125° C. (3.1 g).

IR Spectrum: ν max: 2940, 2865, 1723, 1447, 1383, 1205, 1190, 1180 cm⁻¹.

NMR Spectrum (220 MHz, CDCl₃): τ 1.89 and 1.98 (2H, two singlets, 3- and12-formate protons), τ 4.75 (1H,S,C₁₂ -proton), τ 5.19 (1H,m,C₃-proton), τ 6.71 (1H,m,C₂₃ -proton), τ 6.96 (1H,q,C₂₃ -proton), τ 9.06(3H,S,C₁₉ -protons), τ 9.16 (3H,d,C₂₁ -protons), τ 9.22 (3H,S,C₁₈-protons), τ 7.95-9.15 (24H, steroid nucleus).

(ii)3α,12α-dihydroxy-23-(carboxymethyltelluro)-24-nor-5β-cholane-^(123m) Te

^(123m) -Tellurium (6 mg, 5 mCi) was dissolved in concentratedhydrochloric acid (2 ml) and hydrogen peroxide (100 vol. 2 drops).Tellurium oxide (23 mg inactive) was added and the resulting solutionwas diluted with water (32 ml). Tellurium metal was precipitated usingsulphur dioxide gas, was washed twice with water and then with ethanol,and was finally dried in vacuum.

To tellurium metal (24.6 mg, 5 mCi) in a reaction vessel containing 15ml of liquid ammonia was added Sodium (4.4 mg), the vessel beingconnected to a vacuum manifold and vented to the atmosphere via acarbosorb/charcoal trap. The reaction mixture was stirred for 5 minutesto obtain disodium ditelluride-^(123m) Te and then iodoacetic acid (35.8mg) was added. The ammonia was allowed to evaporate, and traces ofvolatile matter were removed under reduced pressure.

The residue was redissolved in ethanol (20 ml) and dimethylformamide (10ml) and stirred under an atmosphere of nitrogen. Sodium hydroxide (0.1g) in water (3 ml) and dithiothreitol (50 mg) in water (2 ml) wereadded. After 20 minutes 3α,12α-diformoxy-23-iodo-24-nor-5β-cholane indimethyl formamide (2 ml), was added. The reaction mixture was stirredat 60° for 1 hour and at room temperature overnight. The solvents wereevaporated in vacus, and the residue dissolved in chloroform (2 ml) andthen purified by preparative layer chromatography on cellulose (Avicel FButanol, water, acetic acid 60:25:15). The active band, Rf 0.9-0.96 asobserved by autoradiography, was removed from the plate, and extractedinto chloroform. Evaporation of the chloroform yielded a residue of 350μCi (7%).

TLC: Cellulose; (butanol, water, acetic acid 60:25:15). Major component(>95%) - Rf 0.95.

IR Spectrum: ν max: 2950, 2920, 2860, 1725, 1450, 1385, 1125, 1070,1035, 875, 790, 740 cm⁻¹.

(iii) 3α,12α-dihydroxy-23-(carboxymethyltelluro)-24-nor-5β-cholane

This was prepared as in 8 (ii). Tellurium (59 mg), Sodium (11.5 mg),iodoacetic acid (84 mg), Sodium hydroxide (0.2 g) dithiothreitol (100mg) 3α,12α-diformoxy-23-iodo-24-nor-5β-cholane (190 mg) were used.

Yield 30 mg (16%).

IR Spectrum: ν max: 2940, 2860, 1725, 1450, 1385, 1130, 1070, 875, 790cm⁻¹.

NMR Spectrum (CD₃ OD)(220 MHz): τ 6.05 (1H,S,C₁₂ -proton), τ 8.97(3H,d,C₂₁ -protons), τ 9.08 (3H,S,C₁₉ -protons), τ 9.28 (3H,S,C₁₈-protons).

What we claim is:
 1. A method for investigating body function of amammal, comprising introducing a γ-emitting radioactive Se or Telabelled derivative of a bile acid or a bile salt comprising an aminoacid conjugate of a bile acid or a metabolic precursor thereof in to thelive mammal, and after the elapse of a suitable period of timedetermining the distribution of the radioactivity.
 2. A method asclaimed in claim 1 for investigating bowel function of a mammal,comprising orally administering the γ-emitting radioactive Se or Telabelled derivative of a bile acid or salt or metabolic precursorthereof to the live mammal, and after the elapse of a suitable period oftime determining the distribution of the radioactivity.
 3. A method asclaimed in claim 2, wherein the distribution of the radioactivity isdetermined by body counting.
 4. A method as claimed in claim 2, whereinthe distribution of the radioactivity is determined by faecal counting.5. A method as claimed in claim 1 for investigating liver function of amammal, comprising intravenously administering the γ-emittingradioactive Se or Te labelled derivative of a bile acid or salt ormetabolic precursor thereof to the live mammal, and after the elapse ofa suitable period of time determining the distribution of theradioactivity.
 6. A method as claimed in claim 1, wherein the bile acidor salt or metabolic precursor thereof is labelled with selenium-75 ortellurium-123 m.
 7. A method as claimed in claim 1, wherein the bileacid or salt is labelled at the 19-position.
 8. A method as claimed inclaim 1, wherein the bile acid or salt is labelled in the C-17 sidechain.
 9. A method as claimed in claim 8, wherein the labelled bile acidis 3α, 12α-dihydroxy-22-(carboxymethyl-[⁷⁵ Se]seleno)-23,24-bisnor-5β-cholane.